In cardiac myocytes, Ca2+ release from the sarcoplasmic reticulum (SR) is activated via the "Ca2+-induced-Ca2+ release" mechanism. Biochemical, ultrastructural, physiological evidence suggests that clusters of sarcolemmal L-type Ca2+ channels and sarcoplasmic Ca2+ release channels, called ryanodine receptors (RyRs), are functionally coupled in diadic junctions forming discrete Ca2+ release units. Ca2+ influx via L- type Ca2+ channels is the major trigger for the release units, and their local Ca2+ release events had been visualized directly as "Ca2+ sparks". Recently, we have devised a novel confocal microscopic technique to image local "Ca2+ spikes" activated by strong depolarization, and found that local Ca2+ release once initiated, terminates in a use-dependent manner through a mechanism independent of stochastic closing of RyRs, adaptation of RyRs, adaptation of RyRs, or depletion of SR Ca2+. Our objective of this proposal is to apply this novel technique, as well as conventional Ca2+ transient measurement, in conjunction with simultaneous voltage-clamping, to investigate the influences of local Ca2+ interactions on the activation and termination of Ca2+ release, and their modulation by endogenous factors. We hypothesize, first, that there are multiple L-type Ca2+ channels co-activation the spatially discrete, independent Ca2+ release unit, and Ca2+ cross-signaling between neighboring RyRs within a release-units dictates the Ca2+ sensitivity for release activation. Second, we hypothesize that termination of Ca2+ release in intact myocytes is mediated through a Ca2+-dependent inactivation process is originally proposed by Fabiato (1985), and both the activation and inactivation processes are modulated by the RyR accessory protein, called FK506 binding protein (FKBP), as well as the inactivation processes are modulated by the RyR accessory protein, called FK506 binding protein (FKBP), as well as by the phosphorylation- dephosphorylation of RyRs. To test the first hypothesis, we will determine (1) the stoichiometry of functional L-type Ca2+ channels in a release unit, and (2) the Ca2+-dependent cooperativity of Ca2+ release by comparing Ca2+ sensitivities for release activation when inter- or intra- cluster cross-signaling is prohibited. To test the second hypothesis, we will (1) determine the Ca2+ dependence of Ca2+ release termination, 92) examine the effects and mechanisms through which FKBP modulates local Ca2+ release events, and (3) the physiological role of Ca2+/calmodulin-dependent phosphorylation on the activation and inactivation of Ca2+ release. This project will provide important information on the mechanistic control and regulation of cardiac excitation contraction coupling.